1. Field of the Invention
This invention relates generally to attenuated microbes and, more particularly, to novel attenuated bacteria having an RpoS+ phenotype for use as vaccines and delivery vehicles for genes and gene products and to methods for their preparation. This invention is particularly applicable to Salmonella such as Salmonella enterica serotype Typhi (also referred to as Salmonella typhi).
2. Description of the Related Art
Live attenuated Salmonella strains can serve as delivery vehicles for recombinant antigens or other proteins. As antigen carriers, the recombinant Salmonella have been shown to be useful in live vaccines (For review see Curtiss et al. in Essentials of Musocal Immunology, Kagnoff and Kiyono, Eds., Academic Press, San Diego, 1996, pp. 599-611; Doggett and Brown, in Mucosal Vaccines, Kiyono et al., Eds., Academic Press, San Diego, 1996 pp 105-118; see also Hopkins et al. Infect Immun. 63:3279-3286, 1995; Srinavasin et al Vaccines 95, R. N. Chanock et al., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y., p 273-280, 1995).
Ideally, live attenuated vaccine strains should possess a balance between the two properties of attenuation and immunogenicity. Such vaccine strains would not cause any disease or impair normal host physiology or growth, thus being attenuated, and at the same time be able to colonize the intestine and gut associated lymphoid tissue upon oral administration or other lymphoid organs upon administration by some other route so as to be immunogenic. As a practical matter, however, such an ideal balance has not been achieved (Curtiss, in New Generation Vaccines Woodrow and Levine, Eds., Marcel Dekker, Inc., New York, 1990, pp. 161-188). This may be a result of the almost exclusive focusing of efforts in Salmonella vaccine development on improving the attenuation component of strains rather than on producing strains with high immunogenicity.
Work directed toward achieving attenuation in microbes for use in vaccines has utilized attenuating mutations in biosynthetic genes, regulatory genes and/or genes involved in virulence. (See Doggett and Brown, supra). One such regulatory gene which has been mutated as a means for achieving attenuation has been the rpoS gene. The rpoS gene encodes an alternative sigma factor, RpoS, which is known to regulate the stationary phase expression of over 30 genes (for review, see Loewen and Hengge-Aronis, Annu Rev Microbiol 48:53-80, 1994). The rpoS gene has been shown to contribute to the virulence of Salmonella enterica serotype Typhimurium (also referred to as Salmonella typhimurium) in mice by RpoS regulation of chromosomal as well as plasmid-borne genes (Fang et al., Proc Natl Acad Sci 89:11978-11982, 1992; Norel et al., FEBS Microbiol Lett 99:271-276, 1992; Kowarz et al., J Bacteriol 176:6852-6860, 1994). Similarly, RpoS is thought to contribute to the virulence of Salmonella typhi in humans by an action on chromosomal gene determinants of virulence, inasmuch as these microbes do not possess the virulence plasmid present in S. typhimurium (Robbe-Saule et al., FEMS Microbiol Let 126:171-176, 1995; Coynault et al., Mol Microbiol 22:149-160, 1996). Mutant rpoS S. typhimurium strains have been shown to be attenuated (Fang et al, supra) and capable of eliciting protective immunity in mice (Nickerson and Curtiss, Abstracts of the 96th General Meeting of the American Society for Microbiology B-141:179, 1996; Coynault et al., Mol Microbiol 22:149-160, 1996). As a result, it has been suggested that rpoS mutants may be attractive candidates for the development of vaccines (Nickerson and Curtiss, supra).
Attenuated strains of Salmonella typhi have been used as human vaccines against typhoid fever as well as against heterologous antigens when used as recombinant antigen delivery vehicles (Forrest, in CRC Press Inc., 1994, pp. 59-80; Levine et al, in New Generation Vaccines Woodrow and Levine, Eds., Marcel Dekker, Inc., New York, 1990, pp. 269-287). These vaccines based upon Typhi strains have almost exclusively been derived from the Ty2 strain, in particular, Ty21a, which contains a galE mutation along with other mutations. Ty2 and its Ty21a derivative vaccine strain have been shown to be rpoS mutants and this mutation may account, at least in part, for the attenuation seen with Ty21a and with other vaccine strains derived from Ty2 presumably by the down regulation of chromosomal virulence genes controlled by the rpoS gene product. The Ty21a vaccine is typical of vaccines derived from Ty2 in that although being attenuated, the Ty21a vaccine has proven to have low vaccine efficacy, requiring three high doses of approximately 1010 cfu to induce protective immunity in approximately two-thirds of the vaccinated individuals. (Forrest, supra). Thus, there remains a continuing need for Salmonella typhi strains which exhibit not only low virulence, but, also high immunogenicity for use in vaccines suitable for the delivery of a desired gene product to a host.
Other strains of S. typhi have been reported which may, however, have a functional rpoS gene although this was not appreciated at the time of the report. For example, human vaccines have been reported based upon the 27V and ISP1820 strains (Reitman, J Infect Dis 117:101-107, 1967; Levine et al., J Infect Dis 133:424-429, 1976; Tacket et al., Infect Immun 60:536-541, 1992). Neither of these strains contained a recombinant gene nor were they used to deliver a recombinant gene in a vaccine composition.
In a report of recombinant rpoS+ S. typhi, Coynault et al. disclosed the construction of a Ty2 derivative containing a recombinant rpoS gene which gave the microbe an RpoS+ phenotype. However, this Ty2 derivative was used only in a laboratory study and no additional recombinant gene was incorporated nor was there any teaching of the use of this derivative in a vaccine composition.
Finally, the S. typhi strains ISP1820 and ISP1822 (U.S. Pat. Nos. 5,387,744 and 5,294,441 and PCT application WO/9424291) and the S. typhi strain 531Ty (U.S. Pat. No. 4,837,151) have been used to construct derivative vaccine strains. Although the studies reported herein show ISP1820, ISP1822 and 531Ty to be RpoS+, this was not known at the time of these earlier publications. Furthermore, none of these references recognized the importance of the presence of a functional rpoS gene in achieving a high immunogenicity in a vaccine preparation. As a result, these references did not disclose the selection of vaccine strains based upon the presence of an RpoS+ phenotype.
All references cited in this specification either supra or infra are hereby incorporated by reference. The discussion of the references herein is intended to summarize the assertions made by their authors and no admission is made as to the accuracy or pertinency of the cited references or that any reference is material to patentability.
In accordance with the present invention, the inventors herein have succeeded in discovering the critical importance of a functional rpoS gene in Salmonella vaccine strains in that the presence of a functional rpoS gene and an RpoS+ phenotype confers upon the Salmonella the property of high immunogenicity. As a result, when the RpoS+ phenotype is present with one or more inactivating mutations other than a mutation in an rpoS gene, which render the microbe attenuated, a new and advantageous balance of attenuation and high immunogenicity is achieved. This invention is particularly applicable to S. typhi based vaccines, however, it is also applicable to other Salmonella such as S. paratyphi A, B and C as well as to other serotypes of S. enterica such as Typhimurium, Enteritidis, Dublin and Choleraesuis. The invention is also applicable to other bacteria having an rpoS gene, or functional equivalent thereof, that can colonize human tissues, including Shigella, E. coli, and hybrids between such bacteria, such as Salmonella-Shigella hybrids, Salmonella-E. coli hybrids or Shigell-E. coli hybrids.
In one embodiment of the present invention, a method is provided for delivery of a desired gene product to a human. The method comprises selecting a strain of bacteria such as S. typhi on the basis of the strain having (i) an RpoS+ phenotype, (ii) one or more inactivating mutations which render the strain attenuated, and (iii) a recombinant gene encoding the gene product. The selecting step with respect to RpoS+ phenotype can involve, in whole or in part, testing the strain to determine its RpoS phenotype. The strain thus selected is then administered to the human. The one or more inactivating mutations which render the strain attenuated can involve a mutation in one gene or a mutation in each of two or more genes.
The RpoS+ phenotypic activities of the Salmonella or other bacteria can be produced by a chromosomal rpoS gene and/or by a recombinant.gene introduced into the strain. Thus, in another embodiment, the method comprises administering to a human a live attenuated strain of bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) a second recombinant gene encoding the desired product. By recombinant rpoS+ gene or wild-type rpoS gene it is meant that the rpoS gene is capable of producing a functional rpoS gene product. The term recombinant rpoS+ gene is intended to refer to an rpoS gene introduced into a microbe by human intervention and to exclude an rpoS gene transferred from and by a wild-type microbe using a natural means of gene transfer such as conjugation, transduction or transformation, without aid of human intervention.
The attenuated microbes of the present invention contain at least one recombinant gene capable of expressing a desired gene product, which allows their use as carriers or delivery vehicles of the gene product to humans. Examples of gene products deliverable by the microbes of the invention include but are not limited to: antigens, which can be from a human pathogen, or, for use in autoimmune applications, from the human itself, such as, for example, a gamete-specific antigen; enzymes that can synthesize antigens such as polysaccharides, lipoproteins, glycoproteins, and glycolipids; allergens of the human; immunoregulatory molecules; hormones; and pharmacologically active polypeptides. By delivery of the desired gene product it is meant that either the gene product or the polynucleotide, i.e. nucleic acid, either DNA or RNA, encoding the product is delivered to the human. In embodiments in which the attenuated bacteria contains a recombinant rpoS gene, the desired gene product is encoded by a second recombinant gene.
In another embodiment, the present invention provides a method for producing a strain of carrier microbes for delivery of a desired gene product to a human. The method comprises (1) selecting for a strain of S. typhi or other bacteria having an RpoS+ phenotype; (2) producing one or more inactivating mutations in the RpoS+ strain to render the strain attenuated; and (3) introducing into the strain a recombinant gene encoding a desired gene product. The selecting step can involve, in whole or in part, testing the strain to determine its RpoS phenotype. Steps 1-3 can be performed in any order.
In a further embodiment, the present invention involves another method for producing carrier microbes for delivery of a desired gene product to a human. The method comprises generating a live attenuated strain of S. typhi or other bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) a second recombinant gene encoding the desired product.
Another embodiment of the present invention provides a carrier microbe for the delivery of a desired gene product to a human. The microbe comprises a live attenuated strain of S. typhi or other bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) a second recombinant gene encoding the desired product.
In another embodiment a vaccine is provided for immunization of a human. The vaccine comprises a live attenuated strain of S. typhi or other bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) a second recombinant gene encoding the desired product.
The present invention also provides in another embodiment, a genetically engineered cell. By genetically engineered cell reference is made to a cell in which the DNA has been manipulated in vitro by human intervention, for example, by gene splicing, to generate a new combination of genes, to place a given gene or genes under the control of a different regulatory system, to introduce specific mutations into the DNA molecule and the like. The generation of the genetically engineered cells can employ any combination of molecular genetic and classical microbial genetic means to effect construction of the genetically engineered cell.
The genetically engineered cell comprises a live attenuated strain of S. typhi or other bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) a second recombinant gene encoding the desired product. A method is also provided for the preparation of a vaccine comprising mixing the genetically engineered cells with a pharmaceutically acceptable formulation suitable for administration to a human.
The present invention also provides a genetically engineered bacteria, in particular S. typhi, containing a recombinant virulence gene that is regulated by RpoS, or its functional equivalent, in wild-type bacteria and a method for using the genetically engineered bacteria for the delivery of a desired gene product to a human. The recombinant virulence gene is capable of expressing a gene product that facilitates invasion and colonization of any of the gut associated lymphoid tissues (GALT), nasal associated lymphoid tissue (NALT) or the bronchial associated lymphoid tissue (BALT) and the like which can collectively be called the mucosal associated lymphoid tissue (MALT). The genetically engineered S. typhi or other bacteria can be further characterized as having one or more inactivating mutations which render the microbe attenuated as well as a second recombinant gene encoding the desired product.
In still another embodiment, the present invention provides a method for assessing the RpoS phenotype as an indication of the immunogenicity of a bacteria strain, and in particular, of a Salmonella. It is believed that many bacterial strains propagated and maintained under laboratory conditions accumulate ropS mutations. Thus, it would be useful to provide a method for assessing the RpoS phenotype of a Salmonella or other bacteria, particularly for a strain being developed for use in a vaccine. The method comprises determining the RpoS phenotype of the bacteria by assessing characteristics of the microbe regulated by RpoS. An increased immunogenicity is indicated by the presence of an RpoS+ phenotype compared to the immunogenicity of an isogenic strain having an RpoSxe2x88x92 phenotype. The isogenic RpoSxe2x88x92 strain does not exhibit an RpoS+ phenotype, but otherwise has the same genetic background as the test strain.
As noted above, the delivery of a polynucleotide encoding the desired gene product to a human is within the scope of the methods and compositions.of the present invention. Moreover, each of the embodiments above involving methods and compositions based upon microbes having an RpoS+ phenotype are further contemplated to include methods and compositions for the delivery of a gene or portion thereof to the cells of a human. The gene or portion thereof can comprise a eukaryotic expression cassette that contains the genetic information, either DNA or RNA, that is intended to be delivered to cells of the human.
Thus, in one embodiment of the present invention provides methods for delivery of a gene or portion thereof to the cells of a human. One such method comprises selecting a strain of bacteria such as S. typhi on the basis of the strain having (i) an RpoS+ phenotype, (ii) one or more inactivating mutations which render the strain attenuated, and (iii) the gene or portion thereof. The gene or portion thereof can be within a eukaryotic expression cassette. The selecting step with respect to RpoS+ phenotype can involve, in whole or in part, testing the strain to determine its RpoS phenotype. The method can also comprise delivering to cells of a human, a live attenuated strain of bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) the gene or portion thereof. The gene or portion thereof can be within a eukaryotic expression cassette.
The present invention also provides methods for producing a strain of carrier microbes for delivery of a desired gene or portion thereof to a cell of a human. One such method can comprise (1) selecting for a strain of S. typhi or other bacteria having an RpoS+ phenotype; (2) producing one or more inactivating mutations in the RpoS+ strain to render the strain attenuated; and (3) introducing into the strain the gene or portion thereof. The gene or portion thereof can be within a eukaryotic expression cassette. The selecting step can involve, in whole or in part, testing the strain to determine its RpoS phenotype and the steps can be performed in any order. The method can also comprise generating a live attenuated strain of S. typhi or other bacteria having (a) an RpoS+ phenotype, (b) a recombinant rpoS+ gene, (c) one or more inactivating mutations which render said microbe attenuated and (d) the desired gene or portion thereof. The gene or portion thereof can be within a eukaryotic expression cassette.
The bacteria that can be used for delivery of a gene or portion thereof can be an attenuated Salmonella, E. coli or Shigella or Salmonella-Shigella hybrid, Salmonella-E. coli hybrid or Shigella-E. coli hybrid so long as the attenuated bacteria releases the nucleic acid within the target host cell.
Among the several advantages achieved by the present invention, therefore, may be noted the provision of a carrier microbe which is capable of colonizing and delivering a desired gene product or a desired polynucleotide to the gut associated lymphoid tissue if administered orally, to the nasal associated lymphoid tissue if administered intranasally and to other lymphoid organs if administered by other routes; the provision of an efficient and inexpensive method for delivery of a nucleic acid molecule to human cells based upon the use of RpoS+ carrier bacteria cells that release the nucleic acid molecule; the provision of vaccine preparations which are highly immunogenic along with being attenuated; the provision of methods of delivering a desired gene product or polynucleotide to an individual by administering the carrier microbe so as to elicit an immune response; the provision of methods of preparing RpoS+ carrier microbes and vaccines wherein the vaccines are not only attenuated but also have high immunogenicity; and the provision of methods for assessing the immunogenicity of a Salmonella or other bacteria by determining its RpoS phenotype.